Purification of bovine conglutinin using pepsin digestion

This paper describes a new method for the purification of bovine conglutinin based on the relative resistance of this protein to pepsin digestion. First, conglutinin is purified by absorption on yeast, then the preparation is treated with 2% pepsin (w/w) at 4°C for 18 hr, and finally gel filtrated on agarose A5m. The yield is 60–75% and conglutinin thus prepared appears physically, immunochemically and functionally intact. This procedure allows for a rapid production of sufficient amounts of conglutinin for immune complex detection or purification methods.     

Choroidal melanoma with oculodermal melanocytosis in Hispanic patients.

PURPOSE: To describe three Hispanic patients with oculodermal melanocytosis and uveal melanoma. METHOD: Case series. RESULTS: Three Hispanic patients with oculodermal melanocytosis and uveal melanoma underwent enucleation. The diagnosis of choroidal melanoma was confirmed by histopathologic examination. CONCLUSION: In the Hispanic population, uveal melanoma can occur in the presence of oculodermal melanocytosis.     

Altered mRNA expression for brain-derived neurotrophic factor and type II calcium/calmodulin-dependent protein kinase in the hippocampus of patients with intractable temporal lobe epilepsy

The expression of brain-derived neurotrophic factor and the alpha subunit of calcium/calmodulin-dependent protein kinase II mRNA in hippocampi obtained during surgical resections for intractable temporal lobe epilepsy were examined. Both calcium/calmodulin-dependent protein kinase II and brain-derived neurotrophic factor are localized heavily within the hippocampus and have been implicated in regulating hippocampal activity (Kang and Schuman [1995] Science 267:1658-1662; Suzuki [1994] Intl J Biochem 26:735-744). Also, the autocrine and paracrine actions of brain-derived neurotrophic factor within the central nervous system make it a likely candidate for mediating morphologic changes typically seen in the epileptic hippocampus. Quantitative assessments of mRNA levels in epileptic hippocampi relative to autopsy controls were made by using normalized densitometric analysis of in situ hybridization. In addition, correlations between clinical data and mRNA levels were studied. Relative to autopsy control tissue, decreased hybridization to mRNA of the alpha subunit of calcium/calmodulin-dependent protein kinase II and increased hybridization to brain-derived neurotrophic factor mRNA were found throughout the granule cells of the epileptic hippocampus. There also was a significant negative correlation between the duration of epilepsy and the expression of mRNA for brain-derived neurotrophic factor. These results are similar qualitatively to those found in animal models of epilepsy and suggest that chronic seizure activity in humans leads to persistent alterations in gene expression. Furthermore, these alterations in gene expression may play a role in the etiology of the epileptic condition.     

Genetic and environmental control of host-gut microbiota interactions

Genetics provides a potentially powerful approach to dissect host-gut microbiota interactions. Toward this end, we profiled gut microbiota using 16s rRNA gene sequencing in a panel of 110 diverse inbred strains of mice. This panel has previously been studied for a wide range of metabolic traits and can be used for high-resolution association mapping. Using a SNP-based approach with a linear mixed model, we estimated the heritability of microbiota composition. We conclude that, in a controlled environment, the genetic background accounts for a substantial fraction of abundance of most common microbiota. The mice were previously studied for response to a high-fat, high-sucrose diet, and we hypothesized that the dietary response was determined in part by gut microbiota composition. We tested this using a cross-fostering strategy in which a strain showing a modest response, SWR, was seeded with microbiota from a strain showing a strong response, A×B19. Consistent with a role of microbiota in dietary response, the cross-fostered SWR pups exhibited a significantly increased response in weight gain. To examine specific microbiota contributing to the response, we identified various genera whose abundance correlated with dietary response. Among these, we chose Akkermansia muciniphila, a common anaerobe previously associated with metabolic effects. When administered to strain A×B19 by gavage, the dietary response was significantly blunted for obesity, plasma lipids, and insulin resistance. In an effort to further understand host-microbiota interactions, we mapped loci controlling microbiota composition and prioritized candidate genes. Our publicly available data provide a resource for future studies.Studies carried out over the last decade have revealed that gut microbiota contribute to a variety of common disorders, including obesity and diabetes (Musso et al. 2011), colitis (Devkota et al. 2012), atherosclerosis (Wang et al. 2011), rheumatoid arthritis (Vaahtovuo et al. 2008), and cancer (Yoshimoto et al. 2013). The evidence for metabolic interactions is particularly strong, as a large body of data now supports the conclusion that gut microbiota influence the energy harvest from dietary components, particularly complex carbohydrates, and that metabolites such as the short-chain fatty acids produced by gut bacteria can perturb metabolic traits, including adiposity and insulin resistance (Turnbaugh et al. 2006, 2009; Backhed et al. 2007; Wen et al. 2008; Ridaura et al. 2013). Gut microbiota communities are assembled each generation, influenced by maternal seeding, environmental factors, host genetics, and age, resulting in substantial variations in composition among individuals in human populations (Eckburg et al. 2005; Costello et al. 2009; Human Microbiome Project Consortium 2012; Goodrich et al. 2014). Most experimental studies of host-gut microbiota interactions have employed large perturbations, such as comparisons of germ-free versus conventional mice, and the significance of common variations in gut microbiota composition for disease susceptibility is still poorly understood. Furthermore, while studies with germ-free mice have clearly implicated microbiota in clinically relevant traits, it has proven difficult to identify the responsible taxa of bacteria.We now report a population-based analysis of host-gut microbiota interactions in the mouse. One of the issues we explore is the role of host genetics. Although some evidence is consistent with significant heritability of gut microbiota composition, the extent to which the host controls microbiota composition under controlled environmental conditions is unclear. We also examined the role of common variations in gut microbiota in metabolic traits such as obesity and insulin resistance and mapped loci contributing to the abundance of certain microbiota. We performed our study using a resource termed the Hybrid Mouse Diversity Panel (HMDP), consisting of about 100 inbred strains of mice that have been either sequenced or subjected to high-density genotyping (Bennett et al. 2010). The resource has several advantages for genetic analysis as compared to traditional genetic crosses. First, it allows high-resolution mapping by association rather than linkage analysis, and it has now been used for the identification of a number of novel genes underlying complex traits (Farber et al. 2011; Lavinsky et al. 2015; Parks et al. 2015; Rau et al. 2015). Second, since the strains are permanent, the data from separate studies can be integrated, allowing the development of large, publicly available databases of physiological and molecular traits relevant to a variety of clinical disorders (systems.genetics.ucla.edu and phenome.jax.org). Third, the panel is ideal for examining gene-by-environment interactions, since it is possible to examine individuals of a particular genotype under a variety of conditions (Orozco et al. 2012; Parks et al. 2013).     

Long-term regulation of neuronal high-affinity glutamate and glutamine uptake in Aplysia

An increase in transmitter release accompanying long-term sensitization and facilitation occurs at the glutamatergic sensorimotor synapse of Aplysia. We report that a long-term increase in neuronal Glu uptake also accompanies long-term sensitization. Synaptosomes from pleural-pedal ganglia exhibited sodium-dependent, high-affinity Glu transport. Different treatments that induce long-term enhancement of the siphon-withdrawal reflex, or long-term synaptic facilitation increased Glu uptake. Moreover, 5-hydroxytryptamine, a treatment that induces long-term facilitation, also produced a long-term increase in Glu uptake in cultures of sensory neurons. The mechanism for the increase in uptake is an increase in the V(max) of transport. The long-term increase in Glu uptake appeared to be dependent on mRNA and protein synthesis, and transport through the Golgi, because 5,6-dichlorobenzimidazole riboside, emetine, and brefeldin A inhibited the increase in Glu uptake. Also, injection of emetine and 5,6-dichlorobenzimidazole into Aplysia prevented long-term sensitization. Synthesis of Glu itself may be regulated during long-term sensitization because the same treatments that produced an increase in Glu uptake also produced a parallel increase in Gln uptake. These results suggest that coordinated regulation of a number of different processes may be required to establish or maintain long-term synaptic facilitation.     

DNA microarray reveals changes in gene expression of shear stressed human umbilical vein endothelial cells

Using DNA microarray screening (GeneFilter 211, Research Genetics, Huntsville, AL) of mRNA from primary human umbilical vein endothelial cells (HUVEC), we identified 52 genes with significantly altered expression under shear stress [25 dynes/cm(2) for 6 or 24 h (1 dyne = 10 microN), compared with matched stationary controls]; including several genes not heretofore recognized to be shear stress responsive. We examined mRNA expression of nine genes by Northern blot analysis, which confirmed the results obtained on DNA microarrays. Thirty-two genes were up-regulated (by more than 2-fold), the most enhanced being cytochromes P450 1A1 and 1B1, zinc finger protein EZF/GKLF, glucocorticoid-induced leucine zipper protein, argininosuccinate synthase, and human prostaglandin transporter. Most dramatically decreased (by more than 2-fold) were connective tissue growth factor, endothelin-1, monocyte chemotactic protein-1, and spermidine/spermine N1-acetyltransferase. The changes observed suggest several potential mechanisms for increased NO production under shear stress in endothelial cells.